This project studies development of gene therapy vectors tested in their targeting of a variety of cultured cells and in animal models, with the overall goal of better targeting gene transfer into human peripheral blood hematopoietic progenitors (PBHP) as a target for clinical gene therapy to treat inherited immune deficiencies. The specific goals relating to gene therapy were to develop the pre-clinical systems of gene therapy that could then be applied to correct the genetic defect in the X-linked genetic form of chronic granulomatous disease (CGD) and the X-linked form of severe combined immune deficiency (XSCID). Also in collaboration with Dennis Hickstein of the NCI we are also collaborating on studies to develop gene therapy for leukocyte adhesion deficiency (LAD). Specifically, we developed a retrovirus vector producer cell line that secretes high titers of the MFGS vectors containing the gp91phox cDNA and recently developed new methods to concentrate that virus to high titer that we plan in the future to use in the clinic. Although we have previous studies vectors based on both the Human Immunodeficiency Virus (HIV), we have now developed novel lentivirus vectors to correct X-CGD or X-SCID based on the Simian Immunodeficiency Virus of the macaque type (SIV-mac) that very efficiently targets human, non-human primate and dog hematopoietic stem cells. We have conducted and are in the process of conducting large animal studies with these gamma retrovirus and lentivirus vectors. The NOD/SCID immunodeficient mouse will accept grafts of human hematopoietic stem cells. Using the NOD/SCID mouse/human stem cell chimera we demonstrate the full functional correction of 10-20% of human neutrophils arising in this model from the mobilized peripheral blood stem cells of CGD patients transduced with SIV-gp91phox vector. This unprecedented level of gene correction in this model provides the basis for using this lentivector in a future clinical gene therapy trials for CGD. We have also developed RD114 pseudotyped SIV-common gamma chain (gc) vectors to treat XSCID. We also collaborated with Dr. Felsburg from the University of Pennsylvania who has a dog model of XSCID in which we have tested the ability of both our MFGS-gc and SIV-gc vectors to cure this disorder with in vivo gene therapy in this animal model. In vitro we have achieved levels of over 80% marking of dog stem cells using these vector. Specifically, we have used a novel approach for in vivo gene therapy in the XSCID dogs where 3 day old dogs are injected intravenously with corrected gene therapy vector. Using this method, a number of dog treated with either the gamma retrovirus-gc or SIV-gc vectors have achieved long term high level restoration of the immune system. This is a novel and unprecedented demonstration of the feasibility of in vivo gene correction using direct injection of gene therapy vector. In other studies we are examining the role of different growth factors in stimulating CD34+ stem cells to divide and to determine the relationship between entry into the cell cycle, ability to transduce with retrovirus vectors, and the maintenance or loss of long term engrafting potential. These studies are essential to achieving our goal of high levels of gene transfer into long term engrafting stem cell. In other studies we have studied the effects of low dose radiation or chemotherapy on the engraftment of stem cells in animal models. We have demonstrated high levels of engraftment of gene marked cells in mouse and non-human primate animal models using low intensity conditioning regimens consisting of non-ablative levels of total body radiation. Follow up studies are in progress looking at non-ablative chemotherapy regimens instead of using radiation, and preliminary studies suggested that the non-ablative combination of cyclophosphamide and fludarabine can achieve low level (0.3%) prolonged (greater than 1 year)gene transfer marking of blood cells in the primate model. Evidence from human and animal studies of gene therapy suggest that providing an in vivo growth or survival advantage to genetically corrected blood cells can improve the outcome of gene therapy by increasing the percent of corrected cells in the body. One approach to this is to co-express the therapeutic gene (such as the corrective gene for X linked CGD) with a gene that allows for selective enrichment. In studies with collaborators we have explored the use of the methyguanine methyl transferase (MGMT) which protects against alykating agents such as BCNU in a non-human primate model achieving marking rates of up to 20%.